The present invention, in some embodiments thereof, relates to chimeric polypeptides, polynucleotides encoding same, cells expressing same and methods of producing same.
Tumor necrosis factor alpha (TNFα) is an important, pro-inflammatory cytokine mediating the regulation of diverse inflammatory, infectious and immune-related processes and diseases, TNFα being considered the most important mediator responsible for inflammatory pathology.
TNF-alpha is a 17 kD molecular weight protein, initially synthesized as a transmembrane protein arranged in stable trimers, then cleaved by metalloprotease-TNF alpha converting enzyme (TACE) to form the homotrimeric soluble TNF (sTNF) which engages to its cognate receptors (TNFRI, p55 and TNFRII, p75), expressed ubiquitously. The ubiquitous TNF receptors provide the basis for the wide variety of TNF-alpha mediated cellular responses.
TNF-alpha induces a wide variety of cellular responses, many of which result in deleterious consequences, such as cachexia (loss of fat and whole body protein depletion, leading to anorexia, common in cancer and AIDS patients) and septic shock. Elevated secretion of TNF-alpha has been implicated in a variety of human diseases including diabetes, allograft rejection, sepsis, inflammatory bowel diseases, osteoporosis, in many autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, psoriasis, psoriatic arthritis, hypersensitivity, immune complex diseases, and even in malaria, cancer and lung fibrosis.
The biological effect of TNFα is mediated by the two distinct receptors. TNF-alpha receptors, when shed from mononuclear cells, lower the TNF-alpha levels by “mopping up” and acting as natural inhibitors Neutralization of TNFα by specific antibodies and decoy receptors has become a common strategy for regulation of TNFα mediated toxicity.
To date, five protein-based TNFα antagonists have been approved by the US FDA for clinical use: Cimzia (Certolizumab pegol), a TNFmAb Fab′ fragment-PEG conjugate; Remicade (Infliximab), a TNF rmAB; Humira (Adalimumab), a TNF rmAB, Simponi™ (Golimumab), aTNF human monoclonal antibody and etanercept, a fusion protein of soluble 75 kDa TNFα receptors fused to the Fc fragment of human IgG (registered as Enbrel™).
Etanercept is indicated for rheumatoid arthritis (RA) and other arthritic indications such as juvenile idiopathic arthritis (JIA), psoriasis and Ankylosing Spondylitis (AS). Rheumatoid arthritis (RA) is a chronic disease that affects approximately five million people World Wide. Nearly 500,000 patients worldwide across indications are treated with Enbrel. Enbrel sales in 2010 were 7.8 billion dollars and the total anti-TNF market amounted to 24.04 Billion dollars. Clinical trials of Enbrel therapy, current or completed, include such diverse indications as adult respiratory distress syndrome, pemphigus, Alzheimer's disease, Behcet's syndrome, HIV, myocardial infarct, knee joint synovitis, lupus nephritis, lichen planus, systemic amyloidosis, sciatica, vitiligo, chronic fatigue syndrome, anorexia, TMJ, asthma, bronchitis, diabetes, myelodysplastic disease and others.
Enbrel is currently produced in mammalian cells. The safety of biopharmaceuticals has recently come to the forefront for both patients and health care providers due to outbreaks of emerging pathogens, most notably HIV, HCV, Cruezfeld-Jacob's Disease, West Nile Virus and SARS, in multiple regions of the world, emphasizing the risk of pathogen transmission through the use of human- or animal-derived raw materials, such as blood-derived products (serum, plasma cell medium components, etc) in the manufacture of biopharmaceuticals. For example, approximately half (!) of the hemophilia population contracted HIV until identification and screening for the virus became widespread.
Screening and testing have improved recently, reducing the threat of pathogen transmission, but risks still remain from plasma-derived additives during recombinant manufacturing processes. In particular, the risk from unknown pathogens is significant, as these agents may appear in the blood supply in the future and could have a significant impact on safety of mammalian-cell-based biopharmaceuticals. Of particular concern are biopharmaceutical drugs which require repeated, regular administrations, specifically via injection, increasing the cumulative risk to the patient.
However, eliminating animal-derived components from media can significantly alter culture performance as well as post-translational protein modifications. The glycosylation pattern of an antibody molecule can affect its structural integrity, thus influencing its biological function, physicochemical properties and pharmacokinetics, altering both efficacy and safety, particularly immunogenicity. Although no major outbreaks have occurred in recent years, it is still critical to reduce dependence on blood and plasma components in the manufacture of biopharmaceuticals. Conversely, recombinant protein production in mammalian cell culture is unsafe due to xeno contaminations. In 2009 Genzyme was forced to temporarily close its main factory because of viral contamination. It did not restore full supplies of the drugs until 2011. Due to the shortage in the only approved drug for Fabry patients in the US, some people with Fabry disease have suffered heart or kidney problems and one or more may have even died because of the shortage.
Biopharmaceuticals, including modified human proteins, can be produced in transgenic plants in order to address problems of safety, viral infections, immune reactions, production yield and cost. U.S. Pat. No. 6,391,638 and PCT WO2008/135991 teach bioreactor devices for commercial-scale production of recombinant polypeptides from plant cell culture. U.S. Pat. No. 7,951,557, U.S. patent application Ser. Nos. 10/554,387 and 11/790,991 teach construction and expression of nucleic acid vectors for recombinant expression of human proteins in plant cells. PCT WO2007/010533 teaches the expression of recombinant human polypeptides in plant cells, for enteral administration.
Additional background art includes: U.S. Pat. No. 7,915,225 to Finck et al, U.S. patent application Ser. Nos. 13/021,545 and 10/853,479 to Finck et al, U.S. patent application Ser. No. 11/906,600 to Li et al, U.S. patent application Ser. No. 10/115,625 to Warren et al and U.S. patent application Ser. No. 11/784,538 to Gombotz et al.